Alcoholic liver disease is a leading cause of liver failure. This application extends earlier studies supporting a role for hypoxia-inducible factors (HIFs) in the pathogenesis of alcoholic liver disease. HIFs are heterodimeric transcription factors that regulate the cellular response to tissue hypoxia and are overexpressed in many human cancers. We hypothesize that HIF activation underlies the steatotic change seen in the livers of chronically alcohol-fed mice via regulation of a number of downstream target genes. We utilize a mouse model of constitutive, hepatocyte-specific HIF activation to determine whether alcohol-mediated liver injury can be accelerated via this pathway. Increasing evidence suggests that HIF can be activated independent of hypoxia via reactive oxygen radicals. ROS generation by the NADPH oxidase complex is a major consequence of chronic alcohol use in alcoholic liver disease. We postulate that HIF activation in alcoholic liver disease is mediated via ROS generation from NADPH oxidases. Lastly, we propose that these events may be differentially regulated in hepatocytes and Kupffer cells and/or inflammatory cells in the liver. These hypotheses will be tested in the following Specific Aims: AIM1 To evaluate and compare the role of HIF1a and HIF2a in the pathogenesis of alcoholic steatosis by investigating a) Whether an upregulation of HIF1alpha and/or HIF2alpha protein and mRNA in the livers of chronically alcohol-fed mice versus pair-fed controls correlate positively with HIF1 or HIF2 transcription factor activity and expression of downstream targets of HIF1 and HIF2 that are implicated in steatotic change and inflammation;b) Whether constitutively active HIF1a expressed in hepatocytes has an accelerating effect on the pathogenesis of chronic alcohol-mediated liver disease, using a HIF1 transgenic mouse model. Aim 2. To investigate the effect of NADPH oxidases and the cell-specificity in HIF activation in alcohol-induced liver injury by a) Investigating the NADPH oxidase complex in the liver after alcohol feeding in control mice and mice with constitutive active HIF-1 expression b) Testing the effect of alcohol feeding in p47phox deficient mice on steatosis and HIF activation;c) Evaluating whether chronic alcohol feeding differentially regulates HIF1 and HIF2 expression in hepatocytes and Kupffer cells in wild-type and the HIF1a transgenic mouse. d) Dissecting the effect of NADPH oxidase activation by alcohol feeding in mice between hepatocytes and immune cells in bone marrow chimeras generated between p47phox and wild-type mice. Public Health Relevance: Chronic alcohol consumption leads to fat accumulation and inflammation in the liver which over time leads to liver failure. The mechanisms of these alcohol-induced changes in the liver are only partially understood. In this study we will study the role of the hypoxia-inducible factor (HIF), which is a signal transduction molecule involved in fatty liver changes. We will study which specific cell types play a role in HIF-mediated liver damage. In addition, we will also investigate a enzyme system (NADPH) that produces reactive oxygen radicals after alcohol intake as a potential trigger for HIF activation and liver disease. Our studies will define specific therapeutic targets for potential treatment of alcoholic liver disease.